ABSTRACT / PROJECT SUMMARY: Despite the fact that isolates recovered from fungal infections frequently test as sensitive to antifungal drugs in vitro, chronic fungal infections are notoriously hard to treat. The mechanisms behind this resistance are not well understood, though recent studies of bacterial infections and human tumors are starting to acknowledge that heterogeneity within populations may lead to a misleading assessment of drug susceptibility upon testing only one isolate. This proposal aims to use a unique collection of Candida lusitaniae isolates, obtained from chronic fungal infections in subjects with cystic fibrosis, to learn about naturally-occurring heterogeneity in antifungal resistance that develops even in the absence of antifungal treatment. We found a wide range of heterogeneity in drug resistance phenotypes among isolates from three separate subjects. In-depth analysis of whole genome sequences of twenty isolates from one subject revealed heterogeneity in the sequence of MRR1, which encodes a known antifungal resistance regulator. Analysis of mating progeny and constructed mutants, has shown that MRR1 allele type determines fluconazole susceptibility. This proposal aims to use these isolates to investigate some of the outstanding questions regarding Mrr1-mediated drug resistance, including how MDR1 and other Mrr1-regulated transporters contribute to high levels of fluconazole resistance, as well as resistance to other biologically relevant toxins, like bacterially produced phenazines. In addition to a more mechanistic understanding of fluconazole resistance, this proposal also outlines studies aimed to understand why variance in the MRR1 locus was so strongly selected for. I will utilize these isolates to determine if all Mrr1 variants have increased Mrr1 activity and what this would mean in vivo, which could help identify the host, bacterial or pharmaceutically derived molecule(s) which selected for this variance. These studies will use a variety of mutants in both clinical and lab strains in combination with genetic and molecular analysis to identify key components of the Mrr1-regulon that contribute to fluconazole and phenazine resistance in C. lusitaniae. In addition, these studies will provide insight into how chronically-infecting fungal populations develop MRR1 variation in the absence of antifungal treatment leading to differences in drug resistance among isolates. These studies may prompt a reexamination of the way in which chronic fungal infections are viewed and studied, introducing the need to examine multiple isolates from individuals to accurately assess infecting populations and potentially altering treatment strategies to account for isolates with heterogeneous drug resistance harbored within the population.